Self-mixing anticoagulant composition



3,444,093 SELF-MIXING ANTICOAGULANT COMPOSITION Ralph Richheimer, Flushing, N.Y., assignor to Burton Parsons Chemicals, Inc., Washington, D.C., a corporation of Delaware No Drawing. Continuation-impart of application Ser. No. 626,690, Mar. 29, 1967. This application Jan. 29, 1968, Ser. No. 701,131

Int. Cl. A61] 13/00 U.S. Cl. 252380 9 Claims ABSTRACT OF THE DISCLOSURE A self-mixing, enzyme activated, stable blood anticoagulant composition consisting essentially of a dry combination of an anticoagulant with sodium perborate, urea peroxide or an anticoagulant-hydrogen peroxide hydrate. The compositions are tabletted with high density fillers, carried as a coating on a glass or plastic bead or coated on the blood sample container in order to insure complete mixing.

This application is a continuation in part of my copending application Ser. No. 626,690, filed Mar. 29, 1967 and now abandoned.

SUMMARY OF DISCLOSURE This invention relates to blood treatment and more particularly to a method and composition for preventing coagulation of blood. Specifically, the invention relates to an enzyme activated, self-mixing blood additive selected from: (a) at least one anticoagulant in combination with sodium perborate or urea peroxide and (b) at least one anticoagulant hydrogen peroxide hydrate formed by admixing an aqueous solution of hydrogen peroxide and at least one anticoagulant and then drying the slurry formed.

DISCLOSURE To make the many kinds of blood analysis required by modern medical technology, whole blood samples must be obtained. One method which uses a needle and syringe comprises inserting the needle into a blood-carrying vein, withdrawing the plunger of the syringe to draw blood from the vein, and then distributing the blood into test tubes for use in one or more analyses.

Another method employs a test tube in which a vacuum has been formed. The blood carrying vein is pierced with a needle and the vacuum in the test tube is broken by piercing the stopper in the tube with the other end of the needle. When sufficient blood has flowed into the tube to make the desired analysis, the needle is removed from the vem.

In both methods, one or more anticoagulants must be added to the sample and mixed thoroughly therewith immediately to prevent coagulation for most analyses cannot be made properly if even a portion of the sample coagulates. Commonly, one or more of these coagulants have been added to the tubes containing the samples and the tubes shaken to hasten mixing. It would be desirable to have an anticoagulant composition which readily selfmixes with a blood sample upon contact therewith to insure that coagulation does not occur.

Self-mixing compositions per se have been proposed for other systems many times in various chemical arts. However, in the case of blood analysis it is imperative that the self-mix be nonreactive with the blood and further not afiect any subsequent tests. Furthermore, there is often times considerable time lapse between the placement of a self-mixing composition in a sample receiver and the addition in blood sample, such time being sufficient for standard self-mixers such as hydrogen peroxide solutions to lose their oxygen releasing activity.

tates atent It is an object of this invention to provide an effective coagulation preventing composition which rapidly self mixes immediately upon contact with whole blood.

Another object is to provide an efiective coagulation preventing composition which can be used to self mix at least one anticogulant with a blood sample obtained by well-known methods.

A further object of the present invention is to provide a stable composition having long shelf life which readily and rapidly self mixes with a blood upon contacting same.

Another object is to provide a self-mixing coagulation preventive which is harmles to whole blood before, during and after promoting rapid mixture thereof with one or more anticoagulants and which is non-reactive with substances added to blood for analysis.

Other aspects, objects and advantages of the invention are apparent from the disclosure and claims.

Broadly, the invention comprises a coagulation preventing composition which self-mixes with coagulatable whole blood upon contacting same comprising at least one anticoagulant and at least one oxygen providing substance, said substance releasing oxygen through blood enzyme activation and a residue which is armless to coagulatable blood and which is non-reactive with substances added to blood for analysis thereof. The oxygen promotes rapid, thorough mixing of the blood with the one or more anticagulants, but is harmless to the substances of which blood is constituted.

Specifically, the composition of this invention is selected from the group consisting of:

(a) At least one anticoagulant in combination with sodium perborate or urea peroxide, and

(b) At least one anticoagulant-hydrogen peroxide hydrate.

Suitable anticoagulants for the practice of this invention include potassium oxalate, lithium oxalate, sodium oxalate, ammonium oxalate, sodium fiuoride, potassium fluoride, sodium citrate, heparin (sodium) and suitable mixtures thereof dependent upon the blood test to be conducted. The specific anticoagulant for each individual test use is well known to those skilled in the art and will not be repeated here. However, the most desirable anticoagulant is ethylene diamine tetraacetate (di, tri or tetra sodium or potassium), which has been found to be compatible with and non-deleterious to almost every known blood test. A preservative which prevents degradation of the anticoagulant may also be added. Such preservatives are, for example, thymol, etc., and can be present in amounts from about 1% to 25%, based on the weight of the anticoagulant. An extremely minor amount of a dye can also be added to tint the composition for easy identification. For example, Safranine 0 would impart a pink color, while methylene blue, eosine and malachite green would be suitable for their known coloring properties. Usually from about 0.1 to .5 mg./ g. of dye is sufiicient to give the self-mixing composition a characteristic color without effecting ultimate blood hue.

Sufiicient anticoagulant should be present in the composition to prevent coagulation of the entire blood sample upon admixture therewith. Specifically, the amount of anticoagulant should vary from about 1 mg. to 20 mg. or more per cc. of blood sample, dependent upon the specific anticoagulant utilized.

Sodium perborate has been found to be the most preferred self-mix additive for the compositions of the instant invention due to its ready, inexpensive availability in high purity form and the fact that its presence does not effect the outcome of most standard blood tests. Although preferably maintained in the anhydrous state for maximum activity, it can be used in the partial or fully hydrated form, since the reaction producing oxygen from the sodium borate is induced by the enzyme catalase present in the blood. Blood samples containing only the sodium borate are alkaline, but can be rendered more acceptable for use with such tests as hematocrit (determination of red blood cell volume of whole blood), which requires a neutral environment, by either careful control of the particular anticogulant and amount or by addition of a neutralizer to the self-mix composition. Particularly suitable for this latter purpose is barium sulfate, which is not only compatible with the tests, but also acts as a dessicant for the composition before use. Magnesium sulfate and calcium sulfate may be substituted, but they do not appear to act as advantageously as the barium sulfate.

Because it is a stable solid, and leaves a residue normally found in whole blood (namely urea) after admixture therewith, urea peroxide is also a desirable mixing agent of the invention. Only one of the major type of blood analysis cannot be made after using urea peroxide to promote admixture of one or more anticoagulants with whole blood, namely a urea analysis.

Mixtures of the one or more anticoagulants and the sodium perborate or urea peroxide can be made by blending the components in dry form in the desired proportion. The resultant blend may be tabletted or otherwise prepared for convenient handling, as will be explained in greater detail intra.

As mentioned above, an anticoagulant-hydrogen peroxide hydrate is also within the scope of the invention. As used herein, the term anticoagulant hydrogen peroxidate hydrate denotes a stable solid of hydrogen peroxide with at least one anticoagulant. To form this hydrate, one of more anticoagulants are added to sufficient hydrogen peroxide solution to form a thick pasty slurry, whereafter water is removed by evaporation. Generally, from about .1 cc. to 2 cc. of a 30% H solution can be used to dissolve one gram of anticoagulant. As a specific example, 0.75 cc. of a 30% H 0 solution in water was added to 1.2 g. ammonium oxalate and 0.8 g. potassium oxalate to form a slurry. After thorough mixing, this slurry was heated at 37 centigrade until a dry solid formed. (Heating is not necessary, but does expedite the drying step.) A very minute amount of a material such as acetanilid may be added for greater stability of the H 0 Acetanilid contains ammonia and its use should be quite limited since in too great an amount may interfere with the urea determination.

To insure complete mixing of the one or more anticoagulants with the blood sample, the coagulation preventing composition preferably contains from about 0.1% to 25% or more peroxide containing substance based on the weight of anticoagulant present. The exact amount of peroxide may vary dependent upon its form and the anticoagulant utilized, the principal criteria only being that the comopsition contain sufiicient peroxide to insure complete and rapid mixture of blood and anticoagulant.

Solid compositions in accordance with the invention can be carried upon a bead made of inert material such as glass, thermoplastic, coated directly on a blood sample tube or tablet-ted with a high density filler. The purpose of these techniques is to insure thorough mixing of the blood sample, the bead or tablet being of such density as to remain at the bottom of the container after the container is filled with a blood sample, whereby the oxygen released provides thorough mixing of the blood from the bottom to the top.

When utilized on a bead, the size of such a head can conveniently range from .1 in. to .5 in. in diameter. A head in diameter can carry 20 mg. of the composition of the invention and is suitable for use with a cc. sample of blood. A bead A in diameter can carry 40 mg. of the mixture, and will thoroughly mix cc. of blood with the anticoagulant in the composition. The bead is generally coated with the anticoagulant-peroxide blend by combining the composition with a minor amount of gum arabic (U.S.P. white powder). Before combining the gum arabic is boiled for about 2 minutes at 100 C to decompose peroxidase compounds which might otherwise affect the self-mixing properties. After boiling, a thick paste of the gum arabic is made with water and the anticoagulant-peroxide blended therein. The beads are then dipped into this paste whereby, after drying, a hard non-flaking coating is obtained. It will be apparent that other binders, such as gum ghutti, may be utilized, the only criterion being that they be non-blood or anticoagulant reactive and soluble in the blood to release the active coagulants. Similarly, other materials than glass are suitable, such as marble chips, porcelain, and various clays, it being only necessary that the base be of high density and inert. Alternative methods of preparation of the bead include applying the binder initially to the bead and thereafter immersing it in the anticoagulant self-mix blend. This method is preferred with the sodium perborate to eliminate premature breakdown.

These techniques can also be practiced in applying the composition to the inside of the sample container, both precombination and precoating techniques being applicable. If tablets are to be prepared, it is essential that high density fillers be included to insure that the tablet stays at the bottom of the test container.

The following examples demonstrate the effectiveness of the compositions of this invention.

EXAMPLE I Mr glass beads, each weighing about 36 mg, were coated with a mixture of gum arabic in hydrogen peroxide solution prepared by combining 1.270 gm. of gum with 15 cc. of 3% H 0 After partial drying (15 minutesambient temperature) the beads were placed in a homogeneous mixture of 10 g. disodium ethylene diamine tetraacetate and 250 :mg. of sodium perborate, shaken, removed, and allowed to clay in an incubator. Each bead was coated with about 7 mg. of self-mix blend. The H 0 in the binder mix was utilized in order to insure that an oxygen rich atmosphere is present during manufacture.

Two beads were placed in each of a series of test tubes and 5 cc. of blood added thereto. In each tube there was immediate visible indication of oxygen release with effervescent action lasting at least 4 and up to 6 minutes. Upon examination of the samples after 2 hours, there was no evidence of blood coagulation. Standard blood tests were conducted which reproduced identical results to those obtained using the conventional anticoagulant compositions wherein the tubes are inverted.

EXAMPLE II A 5 cc. sample of blood was taken from a mans vein and was immediately added to a test tube containing two glass beads /8 in diameter and carrying 10 mg. of a composition formed initially of:

Potassium oxalate g .8 Ammonium oxalate g 1.2 Urea peroxide g .025 Safranine O mg .5

Upon contact with the blood, the composition immediately began releasing oxygen, and after 5 minutes the effervescent action had subsided. There was no evidence of blood coagulation during this mixing period.

This sample was left open for two hours with no visible dehydration or coagulation. Subsequent standard hematological examination tests were performed and the results showed the blood was unaffected by the anticoagu lant composition.

Identical results were obtained with the anticoagulanthydrogen peroxide hydrate described above.

The compositions of the instant invention will find most utilization in human blood test work but also they will be quite useful in animal work. This is true since many animals have a more rapid coagulation time than humans. The coagulation is so rapid that many times when the specimen is being taken from an animal the 5 blood will have clotted in the tube before the blood taking process is complete. The defined compositions will eliminate this problem.

What is claimed is:

1. A dry self-mixing stable composition for preventing coagulation of coagulatable blood which is nonreactive with the blood and substantially will not affect any subsequent tests, said composition consisting essentially of a member selected from the group consisting of (a) at least one blood compatible anticoagulant which will not prevent the oxygen-providing ingredient to lose its oxygen-releasing activity in combination with sodium perborate or urea peroxide in an amount of about 0.1 to 25% based on the Weight of the anticoagulant and (b) at least one said anticoagulant in an anticoagulant- ...hydrogen peroxide hydrate containing about 0.1 to 2 cc.

of hydrogen peroxide per gram of anticoagulant.

2. The composition of claim 1 wherein the anticoagu lant is selected from the group consisting of potassium oxalate, lithium oxalate, sodium oxalate, ethylene diamine tetraacetate, ammonium oxalate, sodium fluoride, potassium fluoride, sodium citrate, heparin and mixtures thereof.

3. The composition of claim 2 which also contains as a preservative theymol present from about 0.1 to 25% by weight based on the weight of the anticoagulant.

4. The composition of claim 2 wherein the composition contains sodium perborate and the anticoagulant is ethylene diamine tetraacetate.

5. The composition of claim 4 also containing as a neutralizer barium sulfate.

6. The composition of claim 2 wherein the composition containing urea peroxide and the anticoagulant is a blend of potassium oxalate and ammonium oxalate.

7. The composition of claim 1 wherein the anticoagulant-hydrogen peroxide hydrate is a combination of potassium and ammonium oxylates hydrogen peroxide potassium and ammonium oxylates, hydrogen peroxide hydrates.

8. The composition of claim 1 which also contains blood compatible gum arabic, the composition being carried as a coating on an inert sphere about .1 to .5 inch in diameter.

9. The composition of claim 2 which also includes a coloring agent selected from the group consisting of Safranine O, methylene blue, cosine and malachite green in an amount from about 0.1 to 5 mg./ g. of anticoagulant.

LEON D. ROSDOL, Primary Examiner.

T. GLUCK, Assistant Examiner.

U.S. Cl. X.R. 

